Effects Of Different Icroorganisms On Cell Cycle,apoptosis And Telomerase Of Human Umbilical Cord Mesenchymal Stem Cells

Backgrounds and PurposeHuman umbilical cord Wharton's jelly-derived mesenchymal stem cells(HuMSCs)have good low immunogenicity,immunoregulatory properties,self-renewal,multi-lineage differentiation,and tissue regeneration potential,making them a research hotspot of cell therapy.However,studies have shown that aging is an important factor limiting its function and therapeutic performance as a standardized clinical product.Understanding the aging characteristics of human umbilical cord mesenchymal stem cells and exploring the methods of cell regeneration are effective means to overcome their instability.Escherichia coli and Staphylococcus aureus are clinically common pathogenic bacteria.Probiotics can scavenging hydroxyl radicals and superoxide anions in cells,which has potential anti-aging effect and antioxidant effect.The experiment is intended to explore the human umbilical cord mesenchymal stem cells after co-cultured by different bacteria,whether can keep the original stem cell activity and its surface markers,apoptosis,cell cycle and the influence of the role of telomerase activity to study whether it can anti-aging,for further mesenchymal stem cells in vivo,and even between provides the theory basis for clinical application.Materials and Methods1.Isolation,culture and amplification of HuMSCs by tissue block attachment method;2.Probiotics representatives were selected: bacteria-Lactobacillus acidophilus,fungi-boulardii yeast;Pathogenic bacteria were selected as the control group: Gram-positive bacteria-Staphylococcus aureus,Gram-negative bacteria-Escherichia coli;3.Cell phenotype of HuMSCs was detected by flow cytometry.4.Trypan blue staining was used to detect the cell activity of each microflora co-cultured with HuMSCs for 24h;5.Phenotype of HuMSCs 6hours after co-culture with probiotics was detected by flow cytometry;At the same time,flow cytometry was used to detect the apoptotic rate and cell cycle of HuMSCs 6 hours after co-culture with various bacterial communities.6.The m RNA expression level of telomerase was detected by real-time fluorescence quantitative nucleic acid amplification detection system(q PCR),which indirectly reflected the activity of telomerase.Results1.Cell phenotype: HuMSCs expressed mesenchymal stem cell markers CD73,CD90 and CD105,and lacked hematopoietic lineage markers CD45,CD34,CD11 b,CD19 and HLA-DR,which met the standards of the International Society for Cell Therapy(ISCT).There was no difference in cell phenotype between the control group and the probiotic-stimulated HuMSCs.2.Cell activity: in the control group,the activity rate of HuMSCs cultured for 24 h without bacterial flora stimulation was 87.93%;Experimental group: each bacterial community was co-cultured with HuMSCs for 24 hours: the activity rate of HuMSCs in Escherichia coli group was 11.00%;The activity rate of HuMSCs in Staphylococcus aureus group was 16.67%.The activity rate of HuMSCs in Lactobacillus acidophilus group was 87.23%.The activity rate of HuMSCs in Boulardii yeast group was 86.17%.3.Apoptosis: in the control group,the apoptotic rate of HuMSCs cultured for 6 hours without bacterial flora stimulation was 19.1%.In the experimental group,the apoptotic rate of HuMSCs induced by Escherichia coli was 37.6%,and that caused by S.aureus was 27.19%.The apoptotic rate of HuMSCs induced by Lactobacillus acidophilus was 15.89%,and the apoptotic rate of HuMSCs induced by Boulardii yeast was 18.6%.4.Cell cycle: in the control group,the cell cycle after 6 hours of Hum SCs culture without flora stimulation: 21.41% of the cells were in the S+G2+M phase,and 72.4% of the cells were in the G0/G1 phase.After 6 hours of co-culture with HuMSCs,72.5% cells in Escherichia coli group were in G0/G1 phase.Staphylococcus aureus group: 71.1% cells were in G0/G1 phase.In Lactobacillus acidophilus group,72.5% cells were in G0/G1 phase.Boulardii yeast:71.6% cells were in G0/G1 phase.5.Telomerase activity: in the control group,the telomerase activity expression level was 1,in the experimental group,after 6 hours of culture of HuMSCs stimulated by bacteria,the telomerase activity expression level was 1.65 after co-culture of Escherichia coli and HuMSCs,with a statistical difference between the two groups(P < 0.0004).The telomerase activity expression level after co-culture of S.aureus and HuMSCs was 1.54,which was statistically different from that of the control group(P < 0.002).The telomerase activity expression level after co-culture of Boulardii yeast with HuMSCs was 2.69,which was statistically different from that of the control group(P < 0.0002).The telomerase activity expression level after co-culture of Lactobacillus acidophilus with HuMSCs was 1.73,which was statistically different from that of the control group(P < 0.0081).Conclusion1.Probiotics(Lactobacillus acidophilus and Boulardii yeast)can co-culture with HuMSCs and better preserve the cell activity of HuMSCs,while pathogenic bacteria(Staphylococcus aureus and Escherichia coli)cannot.2.Phenotypes of probiotics did not change after co-culture with HUMSCs.3.Probiotics have a positive effect on reducing the apoptosis of HuMSCs.4.Probiotics and pathogenic bacteria had no significant effect on the cell cycle of HuMSCs.5.The telomerase activity of HuMSCs was enhanced after being stimulated by various bacterial communities,suggesting that the stimulation of different bacterial communities in vitro could make the proliferation of HuMSCs become active.